Unintentional injury to nerves is one of the primary causes of morbidity during surgical resection of tumors as well as in other surgeries. Nerve fibers are difficult to distinguish from the surrounding tissues during surgery, making it difficul for surgeons to carefully work around and avoid their injury. Broadly speaking, the goal of the proposed work is to establish third-harmonic generation (THG) microscopy as an approach to visualize, with high sensitivity, nerve fibers intraoperatively. We have previously shown that THG provides a high-sensitivity approach to imaging myelin in the central nervous system of rodents and have used this for in vivo imaging of myelin in the spinal cord. We recently collected preliminary data that shows this approach can highlight peripheral nerves as well. Ultimately, this imaging modality could allow the surgeon to visualize nerve fibers and then work carefully near them, thereby preventing surgical complications associated with nerve injury. We identify three Specific Aims necessary to complete this work. First, we build on our preliminary data and identify the best set of laser and imaging parameters to visualize myelinated peripheral nerve bundles with THG imaging. Second, we assess the effectiveness of THG imaging to spare nerves by comparing the outcome of prostate resection surgeries in rodents done with and without this novel imaging modality. In this Aim, we further build on previous results and examine not just how well nerves are spared when visualized with THG, but also how completely tumors are extracted when they are imaged with two-photon excited fluorescence (2PEF) microscopy. Critically, the laser and imaging systems necessary for THG imaging are completely compatible with 2PEF imaging, allowing these two modalities to work in concert to help a surgeon both maximize tumor extraction and minimize nerve damage. Taken together, these two aims will establish the utility of our approach to nerve imaging, but use in an operating room environment will clearly require development of a less-unwieldy instrument than our current microscopes. Toward this end, in the third Aim, we design an endoscope that is optimized for THG (as well as 2PEF) imaging and could be used in laparoscopic surgeries (such as many prostate cancer resections) in humans. Taken together, this work will establish the utility of THG as a viable imaging modality for use in surgery and will develop the first-generation instrument that could be used in an operating room. If successful, this work could dramatically reduce the post-surgical morbidity of resection surgeries - especially in cases where critical nerves are close to the resection site, such as in the prostate - and therefore improve patient outcomes.